A Study On The Association Between Primary Open-angle Glaucoma In Sichuan And 8q22 Region Rs284489 And Screening For Mutagenic Mutations In Primary Congenital Glaucoma

Primary open-angle glaucoma(POAG)and primary congenital glaucoma(PCG)are important subtypes of primary glaucoma according to the angle of the patient's anatomical structure and age of onset,respectively.Genetic factors play a very important role in the pathogenesis of glaucoma.At present,it has been determined that genes associated with POAG include trabecular mecocillin-induced protein(MYOC),optic neuropathy-inducing protein(optineurin,OPTN),and tryptophan-aspartic acid repeat 36 gene(WD40-Repeat 36,WDR36),etc.As well as some related sites,among which the rs284489 locus in 8q22 in the Caucasian population in the United States was significantly associated with POAG,but there was no correlation analysis between this locus and POAG in the Chinese population.The causative genes of PCG include cytochrome P450 family 1 subfamily B member 1(CYP1B1)and latent transforming growth factor-? Binding Protein 2(LTBP2),MYOC,etc.However,the determinate pathogenic genes are still far from fully explaining all cases.Therefore,exploring unknown pathogenic mutations or genes and in-depth study of its role in the pathogenesis of glaucoma are very important for the clinical diagnosis and therapeutic intervention of glaucoma..This study was divided into two parts: The purpose of the first part of the study was to analyze the association between the rs284489 locus in the 8q22 region and primary open-angle glaucoma in Sichuan Han Chinese population.This study used the Pearson chi-square test to analyze the frequency distribution of rs284489 alleles and genotypes and the correlation with POAG and gender from 894 POAG patients and 994 normal control population in Sichuan.And the results were assessed using Hardy-Weinberg equilibrium detection.Binary logistic regression was used to analyze the relationship between alleles and genetic models with disease,and to correct for covariates such as gender.The test performance was evaluated using PS: Power and Sample Size Calculation(version 3.1.2).All statistical analyses were performed using SPSS 15.0 software(SPSS Inc,Chicago,IL)with using multiple Bonferroni methods.The results showed that the distribution of rs284489 loci in the case group and the control group all met HWE(P>0.05).There was no significant difference in the frequency distribution of minor allele G in rs284489 between the primary open angle glaucoma group and the control group(allele p*=0.94,95%CI=0.83-1.23);for further analysis the relationship between rs284489 and POAG applying of four genetic models,including additive model 1(AG vs AA),additive model 2(GG vs AA),dominant model(GG+AG vs AA),recessive model(GG vs AG + AA)There was no statistically significant difference in frequency distribution between the case group and the control group in the four genetic models of the rs284489 locus(both corrected p#>0.05);gender differences in this study were not associated with polymorphism at the rs284489 locus(corrected)p#=1.00,original 95% CI=0.88-1.14,corrected 95% CI=0.874-1.14).The conclusion is that there is no statistical correlation between the polymorphism of rs284489 locus and the occurrence of primary open-angle glaucoma in Chinese Sichuan Han population.In the second part,398 cases of PCG patients were subjected to full exome sequencing(WES)and sanger sequencing.Two gene mutations were found,c.[143G>A](p.R48H),c.[731G >A](p.R244H)belongs to the guanylate-binding protein X gene(GBPX),because the results of this study have not yet been published,so this paper represents the gene temporarily by GBPX.The protein encoded by this gene is present in most tissues and organs of the human body,including the ciliary body and retina in the eye.The purpose of this part of the study was to analysis the pathogenicity of these two mutations,whether the structure of the protein could be altered,and whether the expression and localization of the mutant protein in the cell were changed.The mutant protein was subjected to pathogenicity analysis,conservation analysis,and tertiary structure prediction,more,mutant and wild-type expression vectors were designed for immunoblotting experiments and cellular immunofluorescence experiments.The experimental results were that c.[143G>A](p.R48H)and c.[731G>A](p.R244H)were maybe deleterious mutations;in the primary structure,the mutated amino acid residues were located in relatively conserved regions.Amino acid changes may be pathogenic;mutated amino acid residues change the structure of secondary structures and the coiling and folding of tertiary structures and p.R48 H no longer bind GNP.The expression levels of both mutant plasmids in the cells were higher than those in the wild-type plasmid,but there was no difference in the expression positions between the mutant plasmids and the wild-type plasmid.The conclusion is that c.[143G>A](p.R48H),c.[731G>A](p.R244H)may be harmful mutations,the mutations change the protein structure and p.R48 H loses the function of binding to GNP,two cases of protein mutations did not change the location within the cell but increased the amount of expression.